Bioactive Nanoengineered Hydrogels for Bone Tissue Engineering: A

ARTICLE

Bioactive Nanoengineered Hydrogels for Bone Tissue Engineering: A Growth-Factor-Free Approach Janet R. Xavier,† Teena Thakur,† Prachi Desai,† Manish K. Jaiswal,† Nick Sears,† Elizabeth Cosgriff-Hernandez,†,‡ Roland Kaunas,† and Akhilesh K. Gaharwar*,†,‡ †

Department of Biomedical Engineering and ‡Department of Materials Science and Engineering, Texas A&M University, College Station, Texas 77843, United States

ABSTRACT Despite bone's impressive ability to heal after

traumatic injuries and fractures, a significant need still exists for developing strategies to promote healing of nonunion defects. To address this issue, we developed collagen-based hydrogels containing two-dimensional nanosilicates. Nanosilicates are ultrathin nanomaterials with a high degree of anisotropy and functionality that results in enhanced surface interactions with biological entities compared to their respective three-dimensional counterparts. The addition of nanosilicates resulted in a 4-fold increase in compressive modulus along with an increase in pore size compared to collagen-based hydrogels. In vitro evaluation indicated that the nanocomposite hydrogels are capable of promoting osteogenesis in the absence of any osteoinductive factors. A 3-fold increase in alkaline phosphatase activity and a 4-fold increase in the formation of a mineralized matrix were observed with the addition of the nanosilicates to the collagen-based hydrogels. Overall, these results demonstrate the multiple functions of nanosilicates conducive to the regeneration of bone in nonunion defects, including increased network stiffness and porosity, injectability, and enhanced mineralized matrix formation in a growth-factor-free microenvironment. KEYWORDS: nanocomposite hydrogels . tissue engineering . synthetic nanosilicates . scaffolds . two-dimensional (2D) nanoparticles . bone regeneration

N

anocomposite hydrogels are emerging biomaterials used for a range of tissue engineering approaches.1
5 Hydrogels are able to mimic some of the physical and chemical properties of the extracellular matrix (ECM) due to their structural similarity to native tissue. By adding different nanoparticles, including graphene, graphene oxide, carbon nanotubes (CNTs), hydroxyapatite (nHAp), bioactive glasses, and calcium phosphate, the mechanical and biological characteristics of nanocomposite hydrogels can be enhanced.6
8 Most of these nanoparticles have been shown to support cell adhesion and proliferation, and only a few have been shown to influence cell differentiation. Recently, we reported two-dimensional (2D) nanosilicates with a high degree of anisotropy and functionality for biomedical applications.9
12 The recent rapid advances in 2D nanoparticle technologies have raised exciting questions about their interactions XAVIER ET AL.

with biological moieties.3
5 The addition of 2D nanosilicates to human mesenchymal stem cells (hMSCs)9 and adipose stem cells (ASCs)10 was shown to induce osteogenic differentiation without the use of dexamethasone, bone morphogenic protein-2 (BMP-2), or other growth factors. Nanosilicate (Laponite, Naþ0.7[(Mg5.5Li0.3)Si8O20(OH)4]
0.7) is a hydrous sodium lithium magnesium silicate. The degradation products of nanosilicates are magnesium, orthosilicic acid, and lithium, which can be easily absorbed by the body and are individually shown to have properties that are potentially useful for bone tissue engineering.13 For example, lithium, an FDA-approved inhibitor of glycogen synthetase kinase-3β (GSK3β), activates Wntresponsive genes by elevating cytoplasmic β-catenin.14,15 Orthosilicic acid (Si(OH)4) promotes collagen type I synthesis and osteoblast differentiation via the Wnt/ β-catenin signaling pathway. The role of VOL. 9

’

NO. 3

’

* Address correspondence to [email protected] (A. K. Gaharwar). Received for review December 31, 2014 and accepted February 12, 2015. Published online February 12, 2015 10.1021/nn507488s C 2015 American Chemical Society

3109–3118

’

2015

3109 www.acsnano.org

XAVIER ET AL.

RESULTS AND DISCUSSION Nanosilicate
Gelatin Interactions. Nanosilicate (Laponite, Naþ0.7[(Mg5.5Li0.3)Si8O20(OH)4]
0.7) is a hydrous sodium lithium magnesium silicate characterized by a high aspect ratio (20
40 nm in diameter and 1 nm in thickness) (Figure 1b). Due to the unique crystal structure and charged surface of nanosilicates, a strong physical interactions between a nanosilicate and polyampholytic gelatin was expected. Gelatin is denatured collagen and mimics components of the native extracellular matrix in structure and chemical composition. To fabricate covalently cross-linked hydrogels, amine groups present on the gelatin backbone were replaced by a methacrylate group to obtain gelatin methacrylate (GelMA) according to previously published reports.21
24 The interactions between a nanosilicate and a prepolymer (GelMA) were investigated using electrophoresis and transmission electron microscopy (TEM). The zeta potential of GelMA and the nanosilicates was observed to be
11.3 ( 0.4 and
28 ( 3 mV, respectively. With the addition of nanosilicates to GelMA, a decrease in zeta potential was observed (Figure 1c). Specifically, the addition of 0.5, 1, and 2% nanosilicates to GelMA resulted in a decrease in the zeta potential of the nanocomposites to
13 ( 2.6,
33.4 ( 2.9, and
34.4 ( 0.9 mV, respectively. The decrease in the zeta potential of nanocomposites indicates that nanosilicates strongly interact with gelatin at nanosilicate concentrations g 1%. Additionally, our earlier results also indicate that nanosilicates are uniformly dispersed within the gelatin network without any aggregation.11 The shear-thinning characteristics of the prepolymer solutions containing GelMA and GelMA
2% nanosilicates were determined by monitoring viscosity at different shear rates at 37 °C (Figure 1d). The prepolymer solution (5 wt % GelMA) was a liquid at 37 °C, and the addition of nanosilicates significantly increased the viscosity of the solution with nanosilicate concentrations higher than 2%, resulting in a semisolid that needed skilled handling with smaller diameter pipets. The unique shape and surface charge distribution on the nanosilicates provide a significantly higher surface-to-volume ratio compared to other types of nanoparticles. Moreover, due to the presence of both positive and negative charge on the nanosilicates, it results in the formation of a house-of-cards structure and exhibits a shear-thinning characteristic.17
19 Covalently Cross-Linked Nanocomposite Hydrogels. We fabricated a covalently cross-linked gelatin hydrogel (Gel) by exposing a prepolymer solution of GelMA to UV radiation in the presence of a photoinitiator. By adding 0.5, 1, and 2 wt % of nanosilicates to GelMA, covalently cross-linked nanocomposite hydrogels were fabricated for physical and biological characterization. The addition of nanosilicates provided a desired combination of tunable structure and bioactivity characteristics. VOL. 9

’

NO. 3

’

3109–3118

’

ARTICLE

Wnt signaling has been implicated in osteogenic differentiation of stem cells.16 Moreover, nanocomposite hydrogels loaded with nanosilicates are biocompatible and biodegradable under in vivo conditions11 and can be used for a range of biomedical and biotechnological applications. Compared to other types of 2D nanoparticles such as graphene or graphene oxide (GO), nanosilicates have been shown to provide enhanced physical, chemical, and biological functionality due to a discotic charged surface, uniform shape, high surface-to-volume ratio, and biocompatible nature.1
5 The presence of both positive and negative charges on the nanosilicate surface results in unique anisotropic interactions between the nanoparticles that generate new unconventional phases at the nano- and microscales.17
19 This allows nanosilicates to interact with anionic, cationic, and neutral polymers to form a physically cross-linked network with shear-thinning characteristics.11,12,20 The uniform shape of nanosilicates compared to GO provides additional control over the interactions between nanoparticles and polymer chains to develop multifunctional biomaterials.3,5 Moreover, the optical transparency of nanosilicates17 in aqueous media compared to GO permits imaging of the subsurface cellular process to design complex tissue structures. In this study, we developed an injectable osteoinductive collagen-based nanosilicate matrix for growth-factor-free bone tissue engineering. Natural hydrogel-forming polymers such as collagen are able to mimic native tissue-like properties due to the presence of cell-binding domains Arg-Gly-Asp (RGD) and can be easily modified to obtain covalently crosslinked hydrogels with native tissue-like mechanical properties.21
24 The unique shape and surface charge of nanosilicates promote strong interactions with a collagen-based matrix (gelatin) to form self-assembled structures, which can dynamically form and break to result in shear-thinning properties. Upon UV exposure, the self-assembled structures of a collagen-based matrix and nanosilicates form a highly cross-linked and vstable network (Figure 1a). We hypothesize that incorporating nanosilicates within a collagen-based matrix will provide control over the physical properties of the hydrogel network due to enhanced interactions between the nanosilicates and gelatin backbone. Moreover, the ability of a collagen-based hydrogel loaded with nanosilicate to promote mineralized matrix formation in a growth-factor-free microenvironment for regeneration of bone is unique. Given that the treatment of bone defects remains one of the largest challenges in musculoskeletal tissue engineering, our nanocomposite hydrogel represents a new material design in the broadly interesting area of growth-factorfree strategies. Overall, these dynamic and bioactive nanocomposite gels show strong promise in bone tissue engineering applications.

3110

2015 www.acsnano.org

ARTICLE Figure 1. Synthesis of nanosilicate-loaded gelatin hydrogels. (a) Schematic representation of fabrication of nanocomposite hydrogels from GelMA (prepolymer) and nanosilicates by covalent cross-linking under UV radiation. (b) TEM image showing the size and morphology of nanosilicates with an average diameter of 20
30 nm. Nanosilicates were uniformly distributed within a nanocomposite network (2% silicate), and no aggregation was observed (arrows indicate nanosilicates). (c) The interactions between silicates and gelatin were investigated by monitoring zeta potential. (d) Viscosity
shear rate of prepolymer solution (GelMA and GelMA loaded with nanosilicates) highlights shear-thinning characteristic of the prepolymer solution loaded with nanosilicates. (e) FTIR spectra of nanocomposite hydrogel's structure showed the presence of a Si
O
Si peak (960 cm
1) and amides in the nanocomposite hydrogel upon cross-linking. (f) The addition of silicate nanoparticles increased the hydrogel porosity due to enhanced interactions with the polymeric chains (inset). The EDX spectra of a covalently cross-linked gelatin hydrogel (Gel) and a nanocomposite hydrogel indicated the presence of Si and Mg in the nanocomposite, but not in Gel.

The presence of nanosilicates within the gelatin network after the cross-linking process was confirmed via infrared (IR) spectra, as shown in Figure 1e. The peak near 960 cm
1 observed in the nanosilicates was due to stretching of Si
OH and Si
O and bending of Si
O
Si bonds present in the nanosilicates. Gelatin showed prominent amine peaks near 1540 and 1650 cm
1. The nanocomposites contained both Si
OH and amine peaks, indicating the presence of both gelatin and nanosilicates. The hydration properties of nanocomposite hydrogels are important to investigate, as the diffusion of nutrients and waste products strongly depends on the hydration degree.25 Moreover, changing the hydration degree can alter cellular infiltration during in vivo implantation. The hydration degree of nanocomposites was quantifed in different medium compositions, including PBS and cell culture media (DMEM and XAVIER ET AL.

DMEM þ 10% FBS) (Figure S1). The addition of nanosilicates reduced the hydration degree, likely due to strong interactions between gelatin and nanosilicates. Performing the assays with PBS at different pH levels or with culture media instead of PBS did not significantly alter the results. The porosity of tissue-engineered scaffolds plays an important role in directing cellular behavior, as the size and distribution of pores within scaffolds dictate the cellular infiltration and distribution of cells throughout the network.25 Gel hydrogels showed a uniform and highly interconnected network, similar to previously published literature.21,26 The addition of nanosilicates to Gel resulted in an increase in pore size of the covalently cross-linked network (Figure 1f and Figure S2). The Gel hydrogel displayed pore sizes between 0.2 and 0.9 μm, which substantially increased to 0.3
1.5 μm with the incorporation of 2% nanosilicates. This finding VOL. 9

’

NO. 3

’

3109–3118

’

3111

2015 www.acsnano.org

ARTICLE Figure 2. Effect of nanosilicate on mechanical stiffness was investigated using uniaxial compression and rheological testing. (a) Optical images showing mechanical toughness of the Gel and nanocomposite hydrogels (2% nanosilicates) after deformation. (b) Nanocomposite hydrogels were subjected to unconfined compression up to 0.90 strain. From the stress
strain curves, the compressive modulus of the hydrogels was calculated from the 0.10
0.20 strain (toe region) (*p < 0.05, **p < 0.01). (c) Nanocomposite hydrogels were subjected to cyclic compression of amplitude ranging from 0.20 to 0.90 strain. (d) Energy dissipation calculated from the observed hysteresis with different strain. Effect of number of cycles on energy dissipation at 0.8 strain from the Gel and nanocomposites with 2% nanosilicates. (e) Oscillatory rheometric analysis indicates frequency-dependent changes in the elastic (G0 ) and loss (G00 ) moduli.

is interesting in that it differs from the existing reports related to most other nanocomposite materials, in which physical reinforcement of the polymeric network by nanoparticles results in smaller pore size.27,28 In the nanocomposites, the increase in the pore size may be attributed to the enhanced interactions between the nanosilicates and the gelatin, which locally condense the polymer fraction to result in the formation of larger voids. The microscopic images confirmed the uniform dispersion of nanoparticles within the polymer network with no visible aggregates. A further analysis with energy-dispersive X-ray spectroscopy (EDS) also indicated the presence of silicon (Si) and magnesium (Mg) in the nanocomposites, indicating their presence in the porous structure (Figure 1f). Nanosilicates Enhance Mechanical Stiffness. Nanosilicates interacted with the polymeric matrix to increase the mechanical stiffness. Figure 2a shows that the Gel hydrogel readily broke when compressed with a sharp XAVIER ET AL.

object, whereas the addition of 2D nanosilicates significantly improved the network stability under stress. To quantify the effects of nanosilicates on the mechanical stiffness, hydrogel samples were subjected to uniaxial compression testing. The Gel hydrogel showed an increase in stress with an increase in strain (Figure 2b). The modulus calculated from the initial linear region (0.1
0.2 mm/mm strain) of the Gel stress
strain curve was observed to be 3.3 ( 0.4 kPa, which is comparable to previously published reports.26,29 The addition of small amounts of nanosilicates showed a significant increase in compressive modulus. For example, the addition of 0.5, 1, and 2% nanosilicates resulted in an increase in modulus to 4.7 ( 0.9, 8.9 ( 2.1, and 12.9 ( 1.3 kPa, respectively. The 4-fold increase in compressive modulus due to the addition of 2% nanosilicates can be attributed to enhanced nanoparticle
polymer interactions. A 10-fold increase in peak tensile stress at 90% strain was also observed upon VOL. 9

’

NO. 3

’

3109–3118

’

3112

2015 www.acsnano.org

XAVIER ET AL.

noncovalent interactions between the nanosilicates and the Gel. Although the exact interactions between the Gel and nanosilicates have yet to be fully established, it can be expected that free amine and carboxylic groups present on the Gel backbone interact with the charged surface of the nanosilicates. Nanosilicates Enhance in Vitro Biomineralization and Physiological Stability. In vitro biomineralization, which involves the deposition of hydroxyapatite in the presence of simulated body fluid (SBF), can be used to evaluate the in vitro osteogenic bioactivity of the hydrogel network.31 When a biomaterial's surface is submerged in SBF, the formation of apatite-like deposits on the biomaterial surface indicates the bone-bonding capability of the hydrogel network. Collagen is one of the major components of bone and induces the nucleation of hydroxyapatite crystals.32 In our study, scanning electron microscopy (SEM) and energydispersive X-ray spectroscopy (EDX) data clearly indicated the formation of mineralized deposits over the surface of both the Gel and nanocomposites (Figure S3a). Images of these materials stained with Alizarin Red S to identify calcium deposition indicated staining intensity increased with increasing nanosilicate concentration (Figure S3b). Alizarin Red S was extracted with acetic acid and quantified by colorimetric detection.33 The results indicate a nanosilicate dosedependent increase of more than 50% in Alizarin Red S staining. These results suggest that nanosilicates provide additional sites for hydroxyapatite nucleation and growth. Gelatin is a biodegradable polymer and is highly susceptible to enzymatic degradation. The enzymatic degradation rates of nanosilicate-reinforced Gel hydrogels were evaluated in vitro under accelerated degradation conditions by using 2.5 units/mL of a collagenase solution (at 37 °C). Gel hydrogels readily degraded in the presence of collagenase, with full dissolution occurring within 10
12 h (Figure S4a and b), consistent with previous reports.21,29,30 Due to the highly interconnected and porous network (Figure S2), the collagenase solution can easily permeate the hydrogel network and degrade the entire network uniformly and rapidly. The addition of nanosilicates to the gelatin network significantly improved the stability of the hydrogel network under enzymatic conditions. The nanocomposite hydrogels containing 0.5, 1, and 2% nanosilicates degraded completely in approximately 50, 70, and 90 h, respectively. By fitting a first-order degradation (one-phase decay) curve to these results, the half-life of the Gel was observed to be 3.4 h, and the addition of 0.5, 1, and 2% nanosilicates improved the high-life of the network to 9.1, 18.5, and 22.9 h (Figure S4c). The more than 6-fold increase in the physiological stability of Gel hydrogels upon addition of 2% nanosilicates is likely due to the strong interactions between the polymer and nanoparticles. VOL. 9

’

NO. 3

’

3109–3118

’

ARTICLE

addition of nanosilicates with the Gel displaying a stress of 16 ( 2.9 kPa and the nanocomposite containing 2% nanosilicates displaying a stresss of 164 ( 30 kPa. To further investigate the effect of nanosilicates on the elastomeric properties of hydrogels, cyclic compression testing was performed consisting of five cycles of loading and unloading. The energy dissipated during cyclic loading was calculated from the area under the stress
strain curves over a range of strain amplitudes (Figure 2c). Hysteresis became apparent for strains above 0.40, indicating absorption of energy during deformation. Consistent with the results from a single loading (Figure 2b), the maximum stress at each strain amplitude was significantly higher for nanocomposites compared to the Gel hydrogels. The energy absorbed during the deformation (90% cyclic compression) of the Gel and nanocomposite containing 2% nanosilicates was 1.5 ( 0.4 and 10.4 ( 2.3 kJ/m3, respectively (Figure 2d). The addition of 2% nanosilicates to the Gel resulted in a greater than 6-fold increase in energy absorbed by the network. The increased energy dissipation is due to the relatively high stress generated with the stiffer nanocomposite. Furthermore, the curves were repeatable after multiple cycles of loading and unloading, demonstrating that no plastic deformation was detected (Figure 2d). Thus, nanocomposites are each highly elastic under high compressive strains. The effects of the addition of nanosilicates to the Gel network on gel rheology were investigated by subjecting the hydrogels to a range of amplitudes of oscillatory shear stress to calculate the elastic storage (G0 ) and loss (G00 ) moduli (Figure 2e). Each hydrogel showed significantly higher G0 compared to G00 at all shear stress amplitudes tested. Consistent with the results of the compression testing, the addition of nanosilicates increased G0 . In contrast, other types of nanoparticles incorporated within Gel hydrogels display minimal increases in modulus. For example, Shin et al. incorporated 5 mg/mL of multiwalled carbon nanotubes functionalized with carboxyl groups within Gel hydrogels,26 and no significant increase in compressive modulus was observed. In a similar study, the addition of graphene oxide into Gel hydrogels did not significantly change the compressive modulus for a given UV exposure time.29 These studies indicated that these nanoparticles act simply as fillers that did not increase the mechanical stiffness of the hydrogel network.26,29 However, covalent cross-linking of nanoparticles to polymer chains can significantly increase stiffness. For example, Cha et al. observed that 3 mg/mL of methacrylated graphene oxide conjugated to the Gel resulted in a 2- to 3-fold increase in stiffness.30 In the current study, the addition of nanosilicates to the Gel increased the stiffness by more than 4-fold through

3113

2015 www.acsnano.org

ARTICLE Figure 3. In vitro cell adhesion, proliferation, and differentiation on Gel and nanocomposite hydrogels. (a) All hydrogels supported initial cell adhesion/spreading (day 3) and proliferation, as determined by live/dead imaging and Alamar Blue assay of cells seeded on hydrogel surfaces. TCPS acted as the positive control. (b) The ALP activity was quantified and normalized with the amount of dsDNA present on days 3, 7, and 14. The addition of nanosilicates significantly upregulated ALP activity in both normal and osteocondutive media. (c) Inorganic calcium deposited by preosteoblast cells in normal and osteoconductive media was identified using Alizarin Red S staining. The optical images show the deposition of the mineralized matrix (stained red) on day 14 (*p < 0.05, **p < 0.01, ***p < 0.001).

The nanosilicate may act as a multifunctional crosslinker that interacts with multiple gelatin chains to potentially mask cleavage sites from collagenases. Nanocomposites Support Cellular Adhesion and Enhance in Vitro Mineralization. The ability to support initial cell adhesion and subsequent proliferation is an important requirement of a tissue-engineered scaffold. Gelatin is a denatured protein that contains RGD groups, which play a significant role in cell adhesion via integrins. MC3T3 preosteoblasts were used to investigate the effect of nanosilicates on initial cell adhesion and proliferation. The cells readily adhered to Gel, and the addition of nanosilicates did not result in any significant influence in initial cell adhesion (Figures 3a and S5a). The cellular morphology indicated that the nanocomposites are cytocompatible and did not elicit any cytotoxic effects. The metabolic activity of preosteoblasts on the scaffold was quantified as a measure of XAVIER ET AL.

cell proliferation over a period of 14 days in both normal and osteoconductive media (Figures 3a and S5b). All nanocomposites supported cellular proliferation, and cells showed metabolic activity over a period of 14 days similar to cells cultured on conventional tissue culture polystyrene (TCPS) dishes. These results support our previous published results indicating that nanosilicates are cytocompatible.9,10 Moreover, subcutaneous implantation of nanosilicate
gelatin showed a locally restricted inflammatory reaction in the host and complete degradation occurred within 28 days under in vivo conditions.11 On the basis of these studies, nanosilicate-based hydrogels can be easily used for biomedical applications. Specific biochemical and physical cues in the ECM play a critical role in regulating cellular processes and their fate. By controlling these cues in the engineered ECM, it is possible to regulate or enhance outcomes VOL. 9

’

NO. 3

’

3109–3118

’

3114

2015 www.acsnano.org

XAVIER ET AL.

normal and osteoconductive media in the absence of any osteogenic factors. To further evaluate the potential of nanosilicatebased hydrogels for tissue engineering, we evaluated the ability to engineer intrinsic structure with a welldefined microenvironment. We engineered microgels (